The additive genetic variance after bottlenecks is affected by the number of loci involved in epistatic interactions.

We investigated the role of the number of loci coding for a neutral trait on the release of additive variance for this trait after population bottlenecks. Different bottleneck sizes and durations were tested for various matrices of genotypic values, with initial conditions covering the allele frequency space. We used three different types of matrices. First, we extended Cheverud and Routman's model by defining matrices of "pure" epistasis for three and four independent loci; second, we used genotypic values drawn randomly from uniform, normal, and exponential distributions; and third we used two models of simple metabolic pathways leading to physiological epistasis. For all these matrices of genotypic values except the dominant metabolic pathway, we find that, as the number of loci increases from two to three and four, an increase in the release of additive variance is occurring. The amount of additive variance released for a given set of genotypic values is a function of the inbreeding coefficient, independently of the size and duration of the bottleneck. The level of inbreeding necessary to achieve maximum release in additive variance increases with the number of loci. We find that additive-by-additive epistasis is the type of epistasis most easily converted into additive variance. For a wide range of models, our results show that epistasis, rather than dominance, plays a significant role in the increase of additive variance following bottlenecks.

Analysis of selectin ligands supporting hematologic malignancy cell interactions with their microenvironment : focus on leukemia and multiple myeloma

CONTEXTE: Les sélectines sont une famille de trois protéines qui règlent la capture et le roulement des leucocytes et qui initient la cascade d'adhésion. Elles contrôlent également la migration des leucocytes en réponse à un stimulus physiologique ou inflammatoire pour atteindre un organe cible. Le rôle des sélectines et des leurs ligands est bien connu dans l'adhésion des leucocytes normaux à l'endothélium; en revanche, la nature des ligands des sélectines exprimés par les cellules leucémiques et le myélome multiple est peu connue. La récente découverte que la E- et la P-sélectine sont exprimées par les cellules endothéliales et du stroma de la moelle osseuse, nous a incité à examiner leur rôle dans les interactions des cellules malignes avec leur environnement médullaire. RÉSULTATS: Les analyses ont été conduites sur les cellules du sang ou de la moelle osseuse prélevées à des patients atteints de leucémie aiguë ou de myélome multiple et sur des lignées cellulaires. Les ligands des sélectines qui ont été identifiés sur les blastes leucémiques ou les plasmocytes, sont « P-selectin glycoprotein ligand-1 » (PSGL-1), CD44, CD43 et l'endoglycan (EGC), ainsi que les saccharides fucosylés sLex et CLA. Nous avons vérifié dans des expériences d'adhésion cellulaire effectuées dans des conditions de flux que ces ligands sont fonctionnels, étant porteurs des sucres mentionnés, et qu'ils sont capables de supporter le roulement cellulaire dépendant des sélectines. De plus, nous avons montré que la liaison de ces ligands génère des signaux intracellulaires favorisant la prolifération et la survie des cellules de myélome. CONCLUSION. Les données présentées ici montrent que la E- et la P- sélectine du microenvironnement médullaire interagissent avec les cellules leucémiques et de myélome multiple, et que ces interactions activent des voies de signalisation contrôlant la prolifération et la survie cellulaire. Ces effets protecteurs sont impliqués dans la persistance de clones cellulaires malins résistant aux traitements et peuvent conduire à la récidive de la maladie. L'inhibition de ces interactions pourrait fournir de nouvelles options thérapeutiques pour le traitement de ces maladies de mauvais pronostic. - BACKGROUND: Selectins are a family of glycoproteins involved in the first steps of the adhesion cascade, tethering and rolling, during which leukocytes sense tissue specific signals and commit the cells to enter in a particular organ or inflammation site. While the role of selectins and their ligands is well established in supporting normal leukocyte adhesion to vascular endothelium, our knowledge of selectin ligands in two hematological malignancies, acute leukemia and multiple myeloma, is incomplete. The recent discovery that E- and P- selectin are also expressed on bone marrow (BM) endothelial and stromal cells, prompted us to investigate a potential role in selectin-mediated interaction of malignant cells with its protective BM microenvironment. RESULTS. Using cells obtained from blood or BM of patients affected by acute myeloid or lymphoblastic leukemia, or multiple myeloma, as well as cell lines, we characterized the expression of selectin ligands on blasts and plasma cells and identified P-selectin glycoprotein ligand-1 (PSGL-1), CD44, CD43 and endoglycan (EGC), as well as sLex/CLA determinants. Rolling assays under flow conditions allowed us to verify that these ligands are functional, i.e. correctly glycosylated and able to support selectin-mediated rolling. Moreover, we demonstrated that these ligands trigger proliferation and pro-survival signals upon engagement on myeloma cells. CONCLUSIONS. Data presented here demonstrate that E- and P-selectin in the BM microenvironment interact with leukemia and myeloma cells, and suggest that they have an impact on proliferation and survival of malignant plasma cells. These protective effects may induce drug resistance in malignant clones, leading to disease relapse. Interfering with these interactions could provide new therapeutic options. - Le corps humain dépend du système immunitaire pour sa protection face aux agressions, notamment des bactéries ou des virus, ou face à une dysfonction de l'organisme. Ce système est composé de plusieurs types cellulaires, regroupés sous le nom de leucocytes, qui participent à son fonctionnement. Ces cellules se développent à partir d'une cellule souche hématopo'iétique commune qui réside dans la moelle osseuse. Comme c'est le cas dans les autres tissus, les cellules du système immunitaire peuvent aussi développer des cancers, appelés tumeurs hématopoïétiques ou tumeurs du sang. Bien que ces maladies puissent être traitées avec succès grâce à de fortes doses de chimiothérapies ou à d'autres moyens comme les greffes, les patients connaissent un fort taux de rechute. La raison de ces récidives est la survie d'une partie des cellules malignes dans la moelle osseuse, où elles reçoivent une protection au traitement par le biais de l'interaction avec d'autres cellules. Les sélectines (E-, P- et L-sélectine) régulent l'interaction des leucocytes avec l'endothélium (la paroi des vaisseaux sanguins), d'autres leucocytes et les plaquettes ; ces interactions surviennent quand les leucocytes atteignent un site d'inflammation ou un organe cible. Dans la moelle osseuse, la E- et la P-sélectine se trouvent sur les cellules de l'endothélium et sur les macrophages, qui sont d'autres leucocytes faisant partie du stroma de la moelle. Elles pourraient être impliquées dans la protection des cellules cancéreuses évoquée plus haut. Les molécules d'adhésion avec lesquelles les sélectines s'associent, autrement dit les ligands des sélectines, sont des glycoprotéines. Ces protéines ont besoin de sucres spécifiques pour acquérir une telle capacité d'adhésion. Dans le cadre de cette thèse, nous avons étudié deux types de cellules extraites du sang et de la moelle osseuse des patients atteints d'une leucémie aiguë (les blastes) ou de myélome multiple (les plasmocytes), et leur capacité à se lier aux sélectines. Nous avons démontré une interaction entre ces cellules malignes et la E- et/ou la P-sélectine, à condition que les ligands soient correctement décorés. De plus, lors que les plasmocytes se lient aux sélectines, une cascade de signaux à l'intérieur des cellules stimule leur prolifération et leur survie. L'ensemble de ces résultats permet l'identification de nouvelles cibles thérapeutiques potentielles de ces hémopathies de mauvais pronostic.

Trichomonas vaginalis and Tritrichomonas foetus: interaction with fibroblasts and muscle cells - new insights into parasite-mediated host cell cytotoxicity

Trichomonas vaginalis and Tritrichomonas foetus are parasitic, flagellated protists that inhabit the urogenital tract of humans and bovines, respectively. T. vaginalis causes the most prevalent non-viral sexually transmitted disease worldwide and has been associated with an increased risk for human immunodeficiency virus-1 infection in humans. Infections by T. foetus cause significant losses to the beef industry worldwide due to infertility and spontaneous abortion in cows. Several studies have shown a close association between trichomonads and the epithelium of the urogenital tract. However, little is known concerning the interaction of trichomonads with cells from deeper tissues, such as fibroblasts and muscle cells. Published parasite-host cell interaction studies have reported contradictory results regarding the ability of T. foetus and T. vaginalis to interact with and damage cells of different tissues. In this study, parasite-host cell interactions were examined by culturing primary human fibroblasts obtained from abdominal biopsies performed during plastic surgeries with trichomonads. In addition, mouse 3T3 fibroblasts, primary chick embryo myogenic cells and L6 muscle cells were also used as models of target cells. The parasite-host cell cultures were processed for scanning and transmission electron microscopy and were tested for cell viability and cell death. JC-1 staining, which measures mitochondrial membrane potential, was used to determine whether the parasites induced target cell damage. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling staining was used as an indicator of chromatin damage. The colorimetric crystal violet assay was performed to ana-lyse the cytotoxicity induced by the parasite. The results showed that T. foetus and T. vaginalis adhered to and were cytotoxic to both fibroblasts and muscle cells, indicating that trichomonas infection of the connective and muscle tissues is likely to occur; such infections could cause serious risks to the infected host.

Inhibitory receptor-mediated regulation of natural killer cells.

Natural killer (NK) cells are capable of directly recognizing pathogens, pathogen-infected cells, and transformed cells. NK cells recognize target cells using approximately 100 germ-line encoded receptors, which display activating or inhibitory function. NK cell activation usually requires the engagement of more than one receptor, and these may contribute distinct signaling inputs that are required for the firm adhesion of NK cells to target cells, polarization, and the release of cytotoxic granules, as well as the production of cytokines. In this article we discuss receptor-mediated mechanisms that counteract NK cell activation. The distinct intracellular inhibitory signaling pathways and how they can dominantly interfere with NK cell activation signaling events are discussed first. In addition, mechanisms by which inhibitory receptors modulate cellular activation at the level of receptor-ligand interactions are described. Receptor-mediated inhibition of NK cell function serves three main purposes: ensuring tolerance of NK cells to normal cells, enabling NK cell responses to aberrant host cells that have lost an inhibitory ligand, and, finally, allowing the recognition of certain pathogens that do not express inhibitory ligands.

Characterization of SKI-1/S1P-mediated processing of Arenavirus glycoprotein precursors

Arenaviruses are rodent-born world-wide distributed negative strand RNA viruses that comprise a number of important human pathogens including Lassa virus (LASV) which causes more than 3 00'000 infections annually in Western Africa. Lymphocytic choriomeningitis virus (LCMV) is the prototypic member of the arenavirus family, which is divided in two major subgroups according to serological properties and geographical distribution, the Old World and New World arenaviruses. The envelope glycoprotein precursors (GPCs) of arenaviruses have to undergo proteolytic processing to acquire biological function and to be incorporated into progeny virions. A cellular enzyme is responsible for this processing: the Subtilisin Kexin Isozyme-1 or Site-1 protease (SKI- 1/S1P). In this thesis we have studied the relationship between SKI-1/S1P and the envelope GPs of arenaviruses. In a first project, we investigated the molecular interactions between SKI-1/SIP and arenavirus GPCs. Using SKI-1/SIP mutants, we confirmed previously published observations locating LCMV GPC and LASV GPC processing in the Late Golgi/TGN and ER/cis-Golgi, respectively. A single mutation in the cleavage site of LCMV was sufficient to re-locate SKI- 1/SIP-mediated processing from the late Golgi/TGN to the ER/cis-Golgi. We then demonstrated that the transmembrane domain, the C-terminal tail and the phosphorylation sites of SKI-1/S1P are dispensable for GPC processing. Additionally we identified a SKI- 1/S1P mutant defective for autoprocessing at site Β, B' that was selectively impaired in processing of viral GPCs but not cellular substrates. We also showed that a soluble variant of SKI-1/SIΡ was unable to cleave envelope GPs at the cell surface when added in the culture medium. This study highlighted a new target for small molecule inhibitors that would specifically impair GPC but not cellular substrate processing. In a second project, we identified and characterized two residues: LASV GPC Y253 and SKI-1/S1P Y285 that are important for the SKI-1/SIP-mediated LASV GPC cleavage. An alignment of GPC sequences revealed a conserved aromatic residue in P7 position in the GPCs of Old World and Clade C of New World arenaviruses. Mutations in GPC at position P7 impaired processing efficiency. In SKI-1/S1P, mutating Y285 into A negatively affected processing of substrates containing aromatic residues in P7, without affecting others. This property could be used to develop specific drugs targeting SKI-1/SIP-mediated cleavage of LASV GPC without affecting cellular substrates. As a third project we studied the role of the SKI-1/SIP-mediated processing and the unusual stable signal peptide (SSP) for the folding and secretion of soluble forms of the ectodomain of LASV and LCMV glycoproteins. We provide evidence that the transmembrane domain and the cytosolic tail are crucial for the stability of the prefusion conformation of arenavirus GP and that the SSP is required for transport and processing of full-length GP, but not the soluble ectodomain per se. Taken together, these results will lead to a better understanding of the complex interactions between arenavirus GPCs and SKI-1/S IP, paving the avenue for the development of novel anti-arenaviral therapeutics. - Les Arenavirus sont des virus à ARN négatif distribués mondialement et portés par les rongeurs. Cette famille de virus comprend des virus hautement pathogènes pour l'homme comme le virus de Lassa (LASV) qui cause plus de 300Ό00 infections par année en Afrique de l'Ouest. Le virus de la chorioméningite lymphocytaire (LCMV) est le représentant de cette famille qui est divisée en deux sous-groupes selon des critères sérologiques et de distributions géographiques: arenavirus du Nouveau et de l'Ancien monde. Les glycoprotéines d'enveloppe de ces virus (GPCs) doivent être clivées pour être incorporées dans le virus et ainsi lui permettre d'être infectieux. Une enzyme cellulaire est responsable de ce clivage : la Subtilisin Kexin Isozyme-1 ou protéase Site-1 (SKI-l/SlP). Dans cette thèse, nous avons étudié la relation entre cette enzyme cellulaire et les GPs des arenavirus. Dans un premier temps, nous avons étudié les interactions moléculaires entre SKI- 1/S1P et GPC. A l'aide de mutants de SKI-l/SlP, nous avons confirmé des résultats précédemment publiés montrant que les glycoprotéines d'enveloppe de LASV sont clivés dans le réticulum endoplasmique/cis-Golgi alors que celles de LCMV sont clivées dans le Golgi tardif/TGN. Une seule mutation dans le site de clivage de la glycoprotéine de LCMV est suffisante pour changer le compartiment cellulaire dans lequel est clivée cette glycoprotéine. Ensuite, nous avons démontré que le domaine transmembranaire, la partie cytosolique C-terminale ainsi que les sites de phosphorylations de cette enzyme ne sont pas indispensables pour permettre le clivage de GPC. De plus, nous avons identifié un mutant de SKI-l/SlP dans lequel Γ autoprocessing au site B,B' est impossible, incapable de cliver GPC mais toujours pleinement fonctionnelle envers ses substrats cellulaires. Nous avons également démontré qu'une forme soluble de SKI-l/SlP ajoutée dans le milieu de culture n'est pas capable de couper GPC à la surface de la cellule. Cette étude a défini une nouvelle cible potentielle pour un médicament qui inhiberait le clivage des glycoprotéines des arenavirus sans affecter les processus normaux de la cellule. Dans un second project, nous avons identifié deux acides aminés, LASV GPC Y253 et SKI-l/SlP Y285, qui sont important pour le clivage de LASV GPC. Un alignement des séquences de clivage des GPCs a montré qu'un résidu aromatique est conservé en position P7 du site de clivage chez tous les arenavirus de l'Ancien monde et dans le clade C des arenavirus du Nouveau monde. Une mutation de cet acide aminée dans GPC réduit l'efficacité de clivage par SKI-l/SlP. Mutation de la tyrosine 285 de SKI-l/SlP en alanine affecte négativement le clivage des substrats contenant un résidu aromatique en position P7 sans affecter les autres. Cette propriété pourrait être utilisée pour le développement de médicaments spécifiques ciblant le clivage de GPC. Finalement, nous avons étudié le rôle du processing accomplit par SKI-l/SlP et du signal peptide pour le pliage et la sécrétion de formes solubles des glycoprotéines de LASV et LCMV. Nous avons montré que le domaine transmembranaire et la partie cytosolique de GP sont crucials pour la stabilité de la conformation pre-fusionnelle des GPs et que SSP est nécessaire pour le transport et le processing de GP, mais pas de son ecto-domaine soluble. En conclusion, les résultats obtenus durant cette thèse permettrons de mieux comprendre les interactions complexes entre SKI-l/SlP et les glycoprotéines des arenavirus, ouvrant le chemin pour le développement de nouveaux médicaments anti-arénaviraux.

Protein-protein interactions at the adrenergic receptors.

The adrenergic receptors are among the best characterized G protein-coupled receptors (GPCRs) and knowledge on this receptor family has provided several important paradigms about GPCR function and regulation. One of the most recent paradigms initially supported by studies on adrenergic receptors is that both βarrestins and G proteincoupled receptors themselves can act as scaffolds binding a variety of proteins and this can result in growing complexity of the receptor-mediated cellular effects. In this review we will briefly summarize the main features of βarrestin binding to the adrenergic receptor subtypes and we will review more in detail the main proteins found to selectively interact with distinct AR subtype. At the end, we will review the main findings on oligomerization of the AR subtypes.

DL4-mediated Notch signaling is required for the development of fetal αβ and γδ T cells.

T-cell development depends upon interactions between thymocytes and thymic epithelial cells (TECs). The engagement of delta-like 4 (DL4) on TECs by Notch1 expressed by blood-borne BM-derived precursors is essential for T-cell commitment in the adult thymus. In contrast to the adult, the earliest T-cell progenitors in the embryo originate in the fetal liver and migrate to the nonvascularized fetal thymus via chemokine signals. Within the fetal thymus, some T-cell precursors undergo programmed TCRγ and TCRδ rearrangement and selection, giving rise to unique γδ T cells. Despite these fundamental differences between fetal and adult T-cell lymphopoiesis, we show here that DL4-mediated Notch signaling is essential for the development of both αβ and γδ T-cell lineages in the embryo. Deletion of the DL4 gene in fetal TECs results in an early block in αβ T-cell development and a dramatic reduction of all γδ T-cell subsets in the fetal thymus. In contrast to the adult, no dramatic deviation of T-cell precursors to alternative fates was observed in the fetal thymus in the absence of Notch signaling. Taken together, our data reveal a common requirement for DL4-mediated Notch signaling in fetal and adult thymopoiesis.

Interactions between feather-degrading bacteria and an avian host : zebra finches (Taeniopygia guttata)

Abstract: Birds harbor a variety of bacteria on their plumage, some of which can degrade feathers in vitro. Whether these keratinolytic bacteria are active on live birds and can effect feather degradation on birds is debatable. The effect of such bacteria on the body condition and behavior of birds, is unknown. Using a community of feather-degrading bacteria (EB), we investigate the interaction between the activity and load of such bacteria, on the morphology, body condition, and behavior of zebra finches (Taeniopygia guttata). In Chapter 2, we find that the elevated loads of such microbes lead to a reduction in the expression of morphological traits, such as male bill color (a sexually selected trait) and uropygial gland volume, without reducing body mass, or evoking a cellular immune response. We also suggest the presence of a carotenoid based defense response in hosts, to such elevated loads of microbes and document a sex-based difference in the source of carotenoids used for such a response. In Chapter 3, we investigated the effect of EB loads on male mate choice of zebra finches, wherein male choice of females with elevated and un-altered bacterial loads, varied with male size. We found that larger males preferred females with higher bacterial load and smaller males preferred females with lower bacterial load. Chapter 4 demonstrates that the presence of melanin in feathers reduces the growth and activity of the community of feather-degrading bacteria (EB) and that the EB community can effect feather degradation in humid conditions, without broth. Additional results also demonstrate that the EB community consists of bacteria that can attach themselves to feathers on live birds and those that can live freely on avian plumage. Finally, chapter 5 demonstrates that the self-maintenance, social and sexual behaviors of birds are implicated in the infection and horizontal transmission of bacteria. It also suggests a linked oral - faecal - genital mode of transmission of pathogens in birds. These results demonstrate that differential loads of normal flora of vertebrate hosts can effect changes in their morphology and behavior. They also shed light on the role of feather-degrading bacteria in the evolution of melanin polymorphism in birds and suggest that bacteria can be active on live birds. This thesis also highlights the importance of social and, sexual behaviors of birds, in epidemiology. Résumé: Les Oiseaux ont dans leur plumage diverses bactéries dont certaines dégradent les plumes in vitro, néanmoins. Il n'est pas clair, au vu de précédentes études, si ces bactéries kératinolytiques sont actives sur des oiseaux vivants, et si celles-ci dégradent effectivement le plumage de leur hôte, L'effet de ces bactéries sur la condition corporelle ainsi que le comportement des oiseaux n'est pas connu. A l'aide d'une communauté de bactéries dégradant les plumes (EB), non pathogènes, nous examinons les interactions entre l'activité et la charge bactérienne sur la morphologie, la condition corporelle et le comportement du diamant mandarins (Taeniopygia guttata). Dans le chapitre 2, nous montrons qu'une charge élevée de ces microbes mène à une réduction de l'expression de certains traits morphologiques, tels que la couleur du bec chez le mâle (un trait soumis à sélection sexuelle), ainsi que le volume de la glande uropygienne, sans qu'il y ait une réduction de la masse corporelle, ni déclenchement d'une réponse immune cellulaire. Nos données suggèrent la présence d'une défense chez l'hôte à des charges élevées de bactéries basée sur la présence de caroténoïdes. Nous montrons, de plus une différence liée au sexe dans la source des caroténoïdes utilisé pour cette réponse. Dans le chapitre 3 nous examinons l'influence de la charge bactérienne EB sur le choix des mâles chez le diamant mandarins. Des femelles avec une charge bactérienne normale et augmentée sont choisies par les mâles et ce choix varie avec la taille des mâles. Nous avons mis en évidence que les grands mâles préfèrent les femelles avec une charge bactérienne plus élevée. Les petits mâles préfèrent les femelles avec une charge bactérienne réduite. Le chapitre 4 démontre que la présence de mélanine dans les plumes réduit la croissance et l'activité de la communauté de bactéries dégradant le plumage (EB), et que cette communauté EB peut dégrader les plumes dans des conditions humides, sans milieu de culture liquide. De plus nous montrons que cette communauté consiste en des bactéries qui peuvent s'attacher sur les plumes d'oiseaux vivants ainsi que des bactéries libres. Pour finir nous montrons dans le chapitre 5 que la maintenance corporelle, l'interaction sociale et le comportement sexuel de ces oiseaux sont impliqués dans l'infection et la transmission horizontale de ces bactéries. Nos données suggèrent une transmission orale-fécale-génitale des pathogènes chez les oiseaux. Ces résultats montrent que des charges différentes de la flore bactérienne habituelle et non pathogène de vertébrés peuvent affecter leur morphologie et leur comportement. Ils éclaircissent également le rôle des bactéries dégradant les plumes dans l'évolution du polymorphisme mélanique chez les oiseaux et suggèrent que ces bactéries peuvent être actives sur des oiseaux vivants. Cette thèse souligne également l'importance du comportement social et sexuel des oiseaux dans l'épidémiologie.

PPAR-mediated activity of phthalates: A link to the obesity epidemic?

The endocrine disruption hypothesis asserts that exposure to small amounts of some chemicals in the environment may interfere with the endocrine system and lead to harmful effects in wildlife and humans. Many of these chemicals may interact with members of the nuclear receptor superfamily. Peroxisome proliferator-activated receptors (PPARs) are such candidate members, which interact with many different endogenous and exogenous lipophilic compounds. More particularly, the roles of PPARs in lipid and carbohydrate metabolism raise the question of their activation by a sub-class of pollutants, tentatively named "metabolic disrupters". Phthalates are abundant environmental micro-pollutants in Europe and North America and may belong to this class. Mono-ethyl-hexyl-phthalate (MEHP), a metabolite of the widespread plasticizer di-ethyl-hexyl-phthalate (DEHP), has been found in exposed organisms and interacts with all three PPARs. A thorough analysis of its interactions with PPARgamma identified MEHP as a selective PPARgamma modulator, and thus a possible contributor to the obesity epidemic.

Thy-1-mediated cell-cell contact induces astrocyte migration through the engagement of αVβ3 integrin and syndecan-4.

Cell adhesion to the extracellular matrix proteins occurs through interactions with integrins that bind to Arg-Gly-Asp (RGD) tripeptides, and syndecan-4, which recognizes the heparin-binding domain of other proteins. Both receptors trigger signaling pathways, including those that activate RhoGTPases such as RhoA and Rac1. This sequence of events modulates cell adhesion to the ECM and cell migration. Using a neuron-astrocyte model, we have reported that the neuronal protein Thy-1 engages αVβ3 integrin and syndecan-4 to induce RhoA activation and strong astrocyte adhesion to their underlying substrate. Thus, because cell-cell interactions and strong cell attachment to the matrix are considered antagonistic to cell migration, we hypothesized that Thy-1 stimulation of astrocytes should preclude cell migration. Here, we studied the effect of Thy-1 expressing neurons on astrocyte polarization and migration using a wound-healing assay and immunofluorescence analysis. Signaling molecules involved were studied by affinity precipitation, western blotting and the usage of specific antibodies. Intriguingly, Thy-1 interaction with its two receptors was found to increase astrocyte polarization and migration. The latter events required interactions of these receptors with both the RGD-like sequence and the heparin-binding domain of Thy-1. Additionally, prolonged Thy-1-receptor interactions inhibited RhoA activation while activating FAK, PI3K and Rac1. Therefore, sustained engagement of integrin and syndecan-4 with the neuronal surface protein Thy-1 induces astrocyte migration. Interestingly we identify here, a cell-cell interaction that despite initially inducing strong cell attachment, favors cell migration upon persistent stimulation by engaging the same signaling receptors and molecules as those utilized by the extracellular matrix proteins to stimulate cell movement.

Tools in pharmacovigilance in psychiatry: therapeutic drug monitoring, pharmacogenetic tests and drug-drug interactions databases

SUMMARYIn order to increase drug safety we must better understand how medication interacts with the body of our patients and this knowledge should be made easily available for the clinicians prescribing the medication. This thesis contributes to how the knowledge of some drug properties can increase and how to make information readily accessible for the medical professionals. Furthermore it investigates the use of Therapeutic drug monitoring, drug interaction databases and pharmacogenetic tests in pharmacovigilance.Two pharmacogenetic studies in the naturalistic setting of psychiatric in-patients clinics have been performed; one with the antidepressant mirtazapine, the other with the antipsychotic clozapine. Forty-five depressed patients have been treated with mirtazapine and were followed for 8 weeks. The therapeutic effect was as seen in other previous studies. Enantioselective analyses could confirm an influence of age, gender and smoking in the pharmacokinetics of mirtazapine; it showed a significant influence of the CYP2D6 genotype on the antidepressant effective S-enantiomer, and for the first time an influence of the CYP2B6 genotype on the plasma concentrations of the 8-OH metabolite was found. The CYP2B6*/*6 genotype was associated to better treatment response. A detailed hypothesis of the metabolic pathways of mirtazapine is proposed. In the second pharmacogenetic study, analyses of 75 schizophrenic patients treated with clozapine showed the influence of CYP450 and ABCB1 genotypes on its pharmacokinetics. For the first time we could demonstrate an in vivo effect of the CYP2C19 genotype and an influence of P-glycoprotein on the plasma concentrations of clozapine. Further we confirmed in vivo the prominent role of CYP1A2 in the metabolism of clozapine.Identifying risk factors for the occurrence of serious adverse drug reactions (SADR) would allow a more individualized and safer drug therapy. SADR are rare events and therefore difficult to study. We tested the feasibility of a nested matched case-control study to examine the influence of high drug plasma levels and CYP2D6 genotypes on the risk to experience an SADR. In our sample we compared 62 SADR cases with 82 controls; both groups were psychiatric patients from the in-patient clinic Königsfelden. Drug plasma levels of >120% of the upper recommended references could be identified as a risk factor with a statistically significant odds ratio of 3.5, a similar trend could be seen for CYP2D6 poor metaboliser. Although a matched case-control design seems a valid method, 100% matching is not easy to perform in a relative small cohort of one in-patient clinic. However, a nested case-control study is feasible.On the base of the experience gained in the AMSP+ study and the fact that we have today only sparse data indicating that routine drug plasma concentration monitoring and/or pharmacogenetic testing in psychiatry are justified to minimize the risk for ADR, we developed a test algorithm named "TDM plus" (TDM plus interaction checks plus pharmacogenetic testing).Pharmacovigilance programs such as the AMSP project (AMSP = Arzneimittelsicherheit in der Psychiatrie) survey psychiatric in-patients in order to collect SADR and to detect new safety signals. Case reports of such SADR are, although anecdotal, valuable to illustrate rare clinical events and sometimes confirm theoretical assumptions of e.g. drug interactions. Seven pharmacovigilance case reports are summarized in this thesis.To provide clinicians with meaningful information on the risk of drug combinations, during the course of this thesis the internet based drug interaction program mediQ.ch (in German) has been developed. Risk estimation is based on published clinical and pharmacological information of single drugs and alimentary products, including adverse drug reaction profiles. Information on risk factors such as renal and hepatic insufficiency and specific genotypes are given. More than 20'000 drug pairs have been described in detail. Over 2000 substances with their metabolic and transport pathways are included and all information is referenced with links to the published scientific literature or other information sources. Medical professionals of more than 100 hospitals and 300 individual practitioners do consult mediQ.ch regularly. Validations with comparisons to other drug interaction programs show good results.Finally, therapeutic drug monitoring, drug interaction programs and pharmacogenetic tests are helpful tools in pharmacovigilance and should, in absence of sufficient routine tests supporting data, be used as proposed in our TDM plus algorithm.RESUMEPour améliorer la sécurité d'emploi des médicaments il est important de mieux comprendre leurs interactions dans le corps des patients. Ensuite le clinicien qui prescrit une pharmacothérapie doit avoir un accès simple à ces informations. Entre autres, cette thèse contribue à mieux connaître les caractéristiques pharmacocinétiques de deux médicaments. Elle examine aussi l'utilisation de trois outils en pharmacovigilance : le monitorage thérapeutique des taux plasmatiques des médicaments (« therapeutic drug monitoring »), un programme informatisé d'estimation du risque de combinaisons médicamenteuses, et enfin des tests pharmacogénétiques.Deux études cliniques pharmacogénétiques ont été conduites dans le cadre habituel de clinique psychiatrique : l'une avec la mirtazapine (antidépresseur), l'autre avec la clozapine (antipsychotique). On a traité 45 patients dépressifs avec de la mirtazapine pendant 8 semaines. L'effet thérapeutique était semblable à celui des études précédentes. Nous avons confirmé l'influence de l'âge et du sexe sur la pharmacocinétique de la mirtazapine et la différence dans les concentrations plasmatiques entre fumeurs et non-fumeurs. Au moyen d'analyses énantiomères sélectives, nous avons pu montrer une influence significative du génotype CYP2D6 sur l'énantiomère S+, principalement responsable de l'effet antidépresseur. Pour la première fois, nous avons trouvé une influence du génotype CYP2B6 sur les taux plasmatiques de la 8-OH-mirtazapine. Par ailleurs, le génotype CYP2B6*6/*6 était associé à une meilleure réponse thérapeutique. Une hypothèse sur les voies métaboliques détaillées de la mirtazapine est proposée. Dans la deuxième étude, 75 patients schizophrènes traités avec de la clozapine ont été examinés pour étudier l'influence des génotypes des iso-enzymes CYP450 et de la protéine de transport ABCB1 sur la pharmacocinétique de cet antipsychotique. Pour la première fois, on a montré in vivo un effet des génotypes CYP2C19 et ABCB1 sur les taux plasmatiques de la clozapine. L'importance du CYP1A2 dans le métabolisme de la clozapine a été confirmée.L'identification de facteurs de risques dans la survenue d'effets secondaire graves permettrait une thérapie plus individualisée et plus sûre. Les effets secondaires graves sont rares. Dans une étude de faisabilité (« nested matched case-control design » = étude avec appariement) nous avons comparé des patients avec effets secondaires graves à des patients-contrôles prenant le même type de médicaments mais sans effets secondaires graves. Des taux plasmatiques supérieurs à 120% de la valeur de référence haute sont associés à un risque avec « odds ratio » significatif de 3.5. Une tendance similaire est apparue pour le génotype du CYP2D6. Le « nested matched case-control design » semble une méthode valide qui présente cependant une difficulté : trouver des patients-contrôles dans le cadre d'une seule clinique psychiatrique. Par contre la conduite d'une « nested case-control study » sans appariement est recommandable.Sur la base de notre expérience de l'étude AMSP+ et le fait que nous disposons que de peux de données justifiant des monitorings de taux plasmatiques et/ou de tests pharmacogénétiques de routine, nous avons développé un test algorithme nommé « TDMplus » (TDM + vérification d'interactions médicamenteuses + tests pharmacogénétique).Des programmes de pharmacovigilances comme celui de l'AMSP (Arzneimittelsicherheit in der Psychiatrie = pharmacovigilance en psychiatrie) collectent les effets secondaires graves chez les patients psychiatriques hospitalisés pour identifier des signaux d'alertes. La publication de certains de ces cas même anecdotiques est précieuse. Elle décrit des événements rares et quelques fois une hypothèse sur le potentiel d'une interaction médicamenteuse peut ainsi être confirmée. Sept publications de cas sont résumées ici.Dans le cadre de cette thèse, on a développé un programme informatisé sur internet (en allemand) - mediQ.ch - pour estimer le potentiel de risques d'une interaction médicamenteuse afin d'offrir en ligne ces informations utiles aux cliniciens. Les estimations de risques sont fondées sur des informations cliniques (y compris les profils d'effets secondaires) et pharmacologiques pour chaque médicament ou substance combinés. Le programme donne aussi des informations sur les facteurs de risques comme l'insuffisance rénale et hépatique et certains génotypes. Actuellement il décrit en détail les interactions potentielles de plus de 20'000 paires de médicaments, et celles de 2000 substances actives avec leurs voies de métabolisation et de transport. Chaque information mentionne sa source d'origine; un lien hypertexte permet d'y accéder. Le programme mediQ.ch est régulièrement consulté par les cliniciens de 100 hôpitaux et par 300 praticiens indépendants. Les premières validations et comparaisons avec d'autres programmes sur les interactions médicamenteuses montrent de bons résultats.En conclusion : le monitorage thérapeutique des médicaments, les programmes informatisés contenant l'information sur le potentiel d'interaction médicamenteuse et les tests pharmacogénétiques sont de précieux outils en pharmacovigilance. Nous proposons de les utiliser en respectant l'algorithme « TDM plus » que nous avons développé.

Human-Computer Interaction with older people through ethnographical lenses: everyday interactions with ICT

Report for the scientific sojourn carried out at the School of Computing of the University of Dundee, United Kingdom, from 2010 to 2012. This document is a scientific report of the work done, main results, publications and accomplishment of the objectives of the 2-year post-doctoral research project with reference number BP-A 00239. The project has addressed the topic of older people (60+) and Information and Communication Technologies (ICT), which is a topic of growing social and research interest, from a Human-Computer Interaction perspective. Over a 2-year period (June 2010-June 2012), we have conducted classical ethnography of ICT use in a computer clubhouse in Scotland, addressing interaction barriers and strategies, social sharing practices in Social Network Sites, and ICT learning, and carried out rapid ethnographical studies related to geo-enabled ICT and e-government services towards supporting independent living and active ageing. The main results have provided a much deeper understanding of (i) the everyday use of Computer-Mediated Communication tools, such as video-chats and blogs, and its evolution as older people’s experience with ICT increases over time, (ii) cross-cultural aspects of ICT use in the north and south of Europe, (iii) the relevance of cognition over vision in interacting with geographical information and a wide range of ICT tools, despite common stereotypes (e.g. make things bigger), (iv) the important relationship offline-online to provide older people with socially inclusive and meaningful eservices for independent living and active ageing, (v) how older people carry out social sharing practices in the popular YouTube, (vi) their user experiences and (vii) the challenges they face in ICT learning and the strategies they use to become successful ICT learners over time. The research conducted in this project has been published in 17 papers, 4 in journals – two of which in JCR, 5 in conferences, 4 in workshops and 4 in magazines. Other public output consists of 10 invited talks and seminars.

Sex-mediated herbivory by galling insects on Baccharis concinna (Asteraceae)

The interaction patterns between the dioecious shrub Baccharis concinna Barroso (Asteraceae) and its speciose galling insect community were studied in southeastern Brazil. Two hypotheses were tested in this study: "the differential reproduction and growth hypothesis" that predicts that male plants present fewer reproductive structures and are larger than female plants; and the 'sex-biased herbivory hypothesis' that predicts that male plants support a larger abundance of insect galls than female plants. Plants did not show sexual dimorphism in growth (= mean leaf number). However, male plants had longer shoots and a lower average number of inflorescences than female plants. These results corroborate the hypothesis that male plants grow more and reproduce less than female plants. No statistically significant difference was found in the number of galls between male and female plants, but a sex by environmental effect on gall number was detected. When each species of galling insect was individually analyzed per population of the host plant, the rates of attack varied between sex and population of the host plant, and they were highly variable among the species of galling insects. These results highlight the importance of the interaction between sex and environment in the community structure of galling insects and indicate that other variables besides host sex may influence the patterns of attack by galling herbivores.

NOTCH1 can initiate NF-κB activation via cytosolic interactions with components of the T cell Signalosome.

T cell stimulation requires the input and integration of external signals. Signaling through the T cell receptor (TCR) is known to induce formation of the membrane-tethered CBM complex, comprising CARMA1, BCL10, and MALT1, which is required for TCR-mediated NF-κB activation. TCR signaling has been shown to activate NOTCH proteins, transmembrane receptors also implicated in NF-κB activation. However, the link between TCR-mediated NOTCH signaling and early events leading to induction of NF-κB activity remains unclear. In this report, we demonstrate a novel cytosolic function for NOTCH1 and show that it is essential to CBM complex formation. Using a model of skin allograft rejection, we show in vivo that NOTCH1 acts in the same functional pathway as PKCθ, a T cell-specific kinase important for CBM assembly and classical NF-κB activation. We further demonstrate in vitro NOTCH1 associates physically with PKCθ and CARMA1 in the cytosol. Unexpectedly, when NOTCH1 expression was abrogated using RNAi approaches, interactions between CARMA1, BCL10, and MALT1 were lost. This failure in CBM assembly reduced inhibitor of kappa B alpha phosphorylation and diminished NF-κB-DNA binding. Finally, using a luciferase gene reporter assay, we show the intracellular domain of NOTCH1 can initiate robust NF-κB activity in stimulated T cells, even when NOTCH1 is excluded from the nucleus through modifications that restrict it to the cytoplasm or hold it tethered to the membrane. Collectively, these observations provide evidence that NOTCH1 may facilitate early events during T cell activation by nucleating the CBM complex and initiating NF-κB signaling.

Tumor necrosis factor-alpha and alphaB-crystallin up-regulation during antibody-mediated demyelination in vitro: a putative protective mechanism in oligodendrocytes.

By using an in vitro model of antibody-mediated demyelination, we investigated the relationship between tumor necrosis factor-alpha (TNF-alpha) and heat shock protein (HSP) induction with respect to oligodendrocyte survival. Differentiated aggregate cultures of rat telencephalon were subjected to demyelination by exposure to antibodies against myelin oligodendrocyte glycoprotein (MOG) and complement. Cultures were analyzed 48 hr after exposure. Myelin basic protein (MBP) expression was greatly decreased, but no evidence was found for either necrosis or apoptosis. TNF-alpha was significantly up-regulated. It was localized predominantly in neurons and to a lesser extent in astrocytes and oligodendrocytes, and it was not detectable in microglial cells. Among the different HSPs examined, HSP32 and alphaB-crystallin were up-regulated; they may confer protection from oxidative stress and from apoptotic death, respectively. These results suggest that TNF-alpha, often regarded as a promoter of oligodendroglial death, could alternatively mediate a protective pathway through alphaB-crystallin up-regulation.